Etanercept Formulations Stabilized with Amino Acids

ABSTRACT

The invention provides stabilized aqueous pharmaceutical etanercept compositions stabilized with an amino acid and suitable for long-term storage of etanercept, methods of manufacture of these compositions, methods of administration, and kits containing same.

FIELD OF THE INVENTION

The present invention relates to aqueous pharmaceutical compositionsstabilized with an amino acid for long-term storage of etanercept,methods of manufacture of the compositions, methods of theiradministration, and kits containing the same. The invention includesetanercept formulations that do not require arginine for stabilization.

BACKGROUND OF THE INVENTION

Polypeptides must often be stored prior to their use. When stored forextended periods, polypeptides are frequently unstable in solution(Manning et al., 1989, Pharm. Res. 6:903-918). To extend their shelflife, additional processing steps have been developed, such as drying,e.g., lyophilization. However, lyophilized pharmaceutical compositionsare less convenient to use.

Typical practices to improve polypeptide stability can be addressed byvarying the concentration of elements with the formulation, or by addingexcipients to modify the formulation (See, for example, U.S. Pat. Nos.5,580,856 and 6,171,586). However, the use of additives can still resultin inactive polypeptides. In addition, in the case of lyophilization,the rehydration step can result in inactivation of the polypeptide by,for example, aggregation or denaturation (Flora et al., 1992, Pharm.Res., 9:33-36; Liu et al., 1991, Biotechnol. Bioeng., 37:177-184).Aggregation of polypeptides is undesirable, as it may result inimmunogenicity (Cleland et al., 1993, Crit. Rev. Therapeutic DrugCarrier Systems, 10:307-377; and Robbins et al., 1987, Diabetes,36:838-845).

Another way to improve polypeptide stability is to use L-arginine at aspecific concentration (U.S. Pat. No. 7,648,702).

One of the polypeptides that is stored for up to two years prior to useis etanercept (Enbrel®, Immunex Corporation), which is a dimeric fusionpolypeptide consisting of the extracellular ligand-binding portion ofthe human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR)linked to the Fc portion of human IgG1. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons(Physicians Desk Reference, 2002, Medical Economics Company Inc.) The Fccomponent of etanercept contains the constant heavy 2 (CH2) domain, theconstant heavy 3 (CH3) domain and hinge region, but not the constantheavy 1 (CH1) domain of human IgG1. An Fc domain can contain one or allof the domains described above. Etanercept is usually produced byrecombinant DNA technology in a Chinese hamster ovary (CHO) mammaliancell expression system.

The present invention provides novel stable liquid formulations ofetanercept that allow its long-term storage.

SUMMARY OF THE INVENTION

The present invention is an aqueous pharmaceutical compositioncomprising etanercept and a stabilizer to inhibit instability,aggregation, misfolding and/or fragmentation of the etanercept, whereinthe stabilizer comprises an amino acid compound selected from the groupconsisting of serine, proline and glutamate.

Various technical terms used in the following discussion are definedbelow in the section entitled “Definitions” and throughout the remainderof the specification.

The stabilized etanercept formulations of the present invention elicitlong term storage stability as characterized by at least one of: (1) SECanalysis at M₃ or T₂ or T₄ (as defined herein) of: monomer contentgreater than about 90%; aggregates content of less than about 3 wt %;and fragment 3 content less than about 5 wt %: and (2) HIC analysis atM₃ or T₂ or T₄ wherein the amount of the composition represented by peak1 of the HIC chromatogram is less than about 3 wt. %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan 80 wt. %; and the amount of the composition represented by peak 3of the HIC chromatogram is less than about 20 wt. %.

In a related aspect the formulations containing proline, serine orglutamate elicits long term storage stability as characterized by atleast one of: (1) SEC analysis at M₃ or T₂ or T₄ of greater than about90 wt. % monomer content; less than about 3 wt. % aggregate(s) content;and less than about 5 wt % fragment 3; and (2) HIC analysis at M₃ or T₂or T₄ wherein the amount of the composition represented by peak 1 of theHIC chromatogram is less than about 3 wt. %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan 80 wt. %; and the amount of the composition represented by peak 3of the HIC chromatogram is less than about 20 wt. %.

In preferred aspects of the stabilized formulations, the formulationselicit long term storage stability as characterized by: an HIC analysisat M₃ or T₂ or T₄ wherein the amount of the composition represented bypeak 2 of the HIC chromatogram is greater than or equal to about 95 wt.%; and wherein, if peak 3 is present on the HIC chromatogram, the amountof the composition represented by peak 3 is less than or equal to about1 wt. %.

The stabilized etanercept formulations as summarized above, optionallyand preferably, contain no arginine, or are essentially free ofarginine.

The formulations of the invention have excellent stability as determinedby SEC (Size Exclusion Chromatography) and HIC (Hydrophobic InteractionChromatography) analysis conducted after one, two or three months ofstorage at 5° C. These formulations are comparable to or better than acommercially available formulation of etanercept, in which arginine is arequired component. Accordingly the present invention is furtherdirected to formulations of etanercept, stabilized with serine, prolineor glutamate, which contain no arginine, or are essentially free ofarginine, and wherein the composition, at M₃ or T₂ or T₄ elicits longterm storage stability that meets one or both of the following criteria:(A) stability comparable to or better than commercially availableetanercept marketed under the trademark Enbrel®, as measured by (i) SECanalysis of the amounts of aggregate(s), monomer and fragment 3 in thecomposition (as defined in the specification) and (ii) HIC analysis ofamounts of material in the composition corresponding to peaks 1, 2 and 3of the HIC chromatogram (as defined in the specification); and (B) anHIC chromatogram in which (i) peak 3 is absent, or essentially absentand (ii) peak 2 represents greater than about 95 wt % of thecomposition; an SEC chromatogram containing essentially no peakcorresponding to aggregate(s); and an SEC chromatogram in which themonomer content represents at least about 95 wt % of the composition.

In one embodiment of the invention in the which the stabilizer isglutamate, the stabilized formulation comprises about 25 to about 75mg/ml etanercept; up to about 150 mM glutamate; less than about 6 wt. %sucrose; optionally up to about 100 mM NaCl; about 1 to about 30 mMsodium phosphate, wherein the composition has a pH of about 6.0 to 6.6;and wherein, optionally, and preferably, the composition contains noarginine or is essentially free of arginine.

A further embodiment of a glutamate stabilized formulation comprisesabout 50 mg/ml etanercept; less than about 150 mM glutamate; about 0 to3% sucrose; about 1 to 30 mM phosphate buffer, optionally up to about100 mM NaCL, and having a pH of about 6.0 to 6.6.

A further glutamate stabilized formulation comprises about 50 mg/mletanercept; about 100 to about 120 mM glutamate; less than about 4 wt. %sucrose, and about 10-30, and preferably about 25 mM phosphate; and hasa pH of about 6.3 to 6.5.

A further glutamate stabilized formulation comprises about 50 mg/mletanercept; about 100 mM glutamate; less than about 2 wt. % sucrose,about 100 mM NaCl; about 10-30, and preferably about 25 mM phosphate;and has a pH of about 6.3 to 6.5.

Yet another glutamate stabilized composition of the invention comprisesabout 50 mg/ml etanercept; about 50 mM glutamate; less than about 2 wt.% sucrose, about 100 mM NaCl; about 10-30, and preferably about 25 mMphosphate; and having a pH of about 6.3 to 6.5.

In a further embodiment in which the stabilizer is serine, thestabilized etanercept composition comprises up to about 150 mM serine;about 0.5 to about 3 wt. % sucrose; about 1 to about 30 mM sodiumphosphate, and wherein the composition has a pH of about 6.0 to 6.6; andwherein, optionally, and preferably, the composition contains noarginine or is essentially free of arginine.

A further embodiment of a serine stabilized etanercept compositioncomprises about 50 mg/ml etanercept; about 100-120 mM serine; about 1wt. % sucrose, and about 10-30, and preferably about 25 mM phosphate;and has a pH of about 6.3 to 6.5.

In a further embodiment in the which stabilizer is proline, the prolinestabilized etanercept composition comprises up to about 150 mM proline;about 0.5 to about 3 wt. % sucrose; about 1 to about 30 mM sodiumphosphate, about 15 to about 100 mM NaCl; and wherein the compositionhas a pH of about 6.0 to 6.6; and wherein, optionally, and preferably,the composition contains no arginine or is essentially free of arginine.

A further embodiment of the proline stabilized etanercept composition ofthe present invention comprises about 50 mM proline, less than about 4wt. % sucrose, about 25 mM NaCl, about 10-30, and preferably about 25 mMphosphate, and having pH of about 6.3 to 6.5.

The etanercept compositions of the invention further afford the abilityto provide formulations which contain acceptable levels of subvisibleparticles. Accordingly, the invention is further directed to serine,proline or glutamate stabilized etanercept formulations having, at M₃ orT₂ or T₄, no more than, on average, about 10,000 subvisible particlesper mL having a size greater than 5 μm. Subvisible particles can bemeasured in a known manner using FlowCam analysis.

The stabilized etanercept composition of the present invention arefurther characterized by: (a) an SEC analysis at M₃ or T₂ or T₄ ofgreater than about 90 wt % monomer content; and less than about 3 wt %aggregate(s) content; and (b) an HIC analysis at M₃ or T₂ or T₄ whereinthe amount of the composition represented by peak 1 of the HICchromatogram is less than about 3 wt %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than 80 wt %;and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 20 wt %.

The stability of the formulations may be further characterized in thatthe compositions exhibit an HIC analysis at M₃ or T₂ or T₄ wherein theamount of the composition represented by peak 1 of the HIC chromatogramis less than about 1%; the amount of the composition represented by peak2 of the HIC chromatogram is greater than about 95 wt %; and the amountof the composition represented by peak 3 of the HIC chromatogram is lessthan about 3 wt %.

In a further embodiment in which the stabilizer is glutamate, thestability of the etanercept formulation may be characterized by an SECanalysis at M₃ or T₂ or T₄ of greater than about 97 wt. % monomercontent and less than about 1 wt % aggregate(s) content; and an HICanalysis at M₃ or T₂ or T₄ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 3 wt %;the amount of the composition represented by peak 2 of the HICchromatogram is greater than about 82 wt %; and the amount of thecomposition represented by peak 3 of the HIC chromatogram is less thanabout 15 wt %. These characteristics are attainable without need forarginine as a stabilizer.

In a further embodiment in which the stabilizer is serine, thestabilized etanercept formulation is characterized by: an SEC analysisat M₃ or T₂ or T₄ of greater than about 97 wt. % monomer content andless than about 1 wt % aggregate(s) content; an HIC analysis at M₃ or T₂or T₄ wherein the amount of the composition represented by peak 1 of theHIC chromatogram is less than about 4 wt %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan about 82 wt %; and the amount of the composition represented bypeak 3 of the HIC chromatogram is less than about 15 wt %. Thesecharacteristics are attainable without need for arginine as astabilizer.

Preferred stabilized composition of the invention exhibit an HICanalysis at M₃ or T₂ or T₄ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 2% andpreferably less than about 1%; the amount of the composition representedby peak 2 of the HIC chromatogram is greater than about 95 wt. % andpreferably greater than about 97%; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 1 wt %,and preferably 0 to 1%. These characteristics are attainable withoutneed for arginine as a stabilizer.

As differentiated from commercially available etanercept provided in anarginine-containing formulation, we found it surprising, in light ofU.S. Pat. No. 7,648,702, that the formulation embodiments of etanerceptdescribed and exemplified herein do not require arginine for long-termstabilization, although arginine may still be added if desired. Theability to provide etanercept formulations stabilized without argininerepresents a potentially significant benefit to the health care systemby providing patients and health care providers with alternativeformulations of storage stable etanercept that may become available atlower cost compared with present commercial etanercept formulation(i.e., Enbrel®) that require arginine for stabilization.

As used herein the term “instability” or like terms denotes the tendencyof the etanercept monomer to undergo a variety of undesiredtransformations during storage. Such transformations include theformation of oligomers and high molecular weight aggregate(s)(hereinafter terms “aggregate(s)” in which multiple copies of theessentially intact etanercept monomer become irreversibly associatedwith one another through a variety of non-covalent attractions (e.g.,electrostatic interactions.) Undesired transformations during storagemay also include degradation of the etanercept monomer to smallerfragments and/or clipped species. Ideally, a formulation of etanerceptshould minimize, to the greatest extent possible, the tendency of theformulation to result, during storage, in the formation of aggregates,misfolded protein, oligomers and/or fragments of etanercept. Animportant benefit resulting from the ability to reduce formation ofunwanted aggregates or fragments is a reduction in the potentialtoxicity and/or immunogenicity of the drug.

The etanercept formulation of the present invention which is optionallyand preferably free, or essentially free of arginine. The term“essentially free of arginine” is intended to mean that arginine, evenif present, is not contributing to the stabilization of the etanerceptmonomer in the formulation to such an extent that a person skilled inthe art would judge its presence beneficial or necessary from astabilization standpoint.

These and other aspects will become apparent from the followingdescription although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are now described in detail. Asused in the description and throughout the claims, the meaning of “a”,“an”, and “the” includes plural reference unless the context clearlydictates otherwise. Also, as used in the description and throughout theclaims, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Additionally, some terms used in thisspecification are more specifically defined below.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. Synonyms for certain termsare provided. A recital of one or more synonyms does not exclude the useof other synonyms. The use of examples anywhere in this specificationincluding examples of any terms discussed herein is illustrative only,and in no way limits the scope and meaning of the invention or of anyexemplified term. The invention is not limited to the variousembodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

“Around,” “about” or “approximately” shall generally mean within 20percent, within 10 percent, within 5, 4, 3, 2 or 1 percent of a givenvalue or range. Numerical quantities given are approximate, meaning thatthe term “around,” “about” or “approximately” can be inferred if notexpressly stated.

The term “etanercept” or “etanercept monomer” or “monomer” is synonymouswith Enbrel®. It refers to a polypeptide which is a dimeric fusionpolypeptide consisting of the extracellular ligand-binding portion ofthe human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR)linked to the Fc portion of human IgG1. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons.For the purposes of the present application, the term “etanercept” alsoencompasses etanercept with minor modifications in the amino acidstructure (including deletions, additions, and/or substitutions of aminoacids) which do not significantly affect the function, potency, oravidity of etanercept. The term “etanercept” encompasses all forms andformulations of Enbrel®, including but not limited to concentratedformulations, injectable ready-to-use formulations; formulationsreconstituted with water, alcohol, and/or other ingredients, and others.

The term “monomer” as used herein is intended to mean the dimericetanercept fusion protein referenced above.

The term “serine” refers to an amino acid whose codons are UCU, UCC,UCA, UCG, AGU, and AGC

The term “proline” refers to an α-amino acid whose codons are CCU, CCC,CCA, and CCG.

The term “glutamate” refers to the deprotonated form or salt of theα-amino acid, glutamic acid (Glu). For the purposes of this application,the term “glutamate” also encompasses glutamic acid itself.

The term “sugar” refers to monosaccharides, disaccharides, andpolysaccharides. Examples of sugars include, but are not limited to,sucrose, trehalose, dextrose, and others.

The term “polyol” refers to an alcohol containing multiple hydroxylgroups. Examples of polyols include, but are not limited to, mannitol,sorbitol, and others.

The term “long-term storage” is understood to mean that thepharmaceutical composition can be stored for three months or more, forsix months or more, and preferably for one year or more. Long-termstorage is also understood to mean that the pharmaceutical compositionis stored either as a liquid at 2-8° C., or is frozen, e.g., at −20° C.,or colder. It is also contemplated that the composition can be frozenand thawed more than once.

The term “stable” or “stabilized” with respect to long-term storage isunderstood to mean that etanercept contained in the pharmaceuticalcompositions does not lose more than 20%, or more preferably 15%, oreven more preferably 10%, and most preferably 5% of its activityrelative to activity of the composition at the beginning of storage.

The term “mammal” includes, but is not limited to, a human.

The term “pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material,formulation auxiliary, or excipient of any conventional type. Apharmaceutically acceptable carrier is non-toxic to recipients at thedosages and concentrations employed and is compatible with otheringredients of the formulation.

The term “composition” refers to a mixture that usually contains acarrier, such as a pharmaceutically acceptable carrier or excipient thatis conventional in the art and which is suitable for administration intoa subject for therapeutic, diagnostic, or prophylactic purposes. It mayinclude a cell culture in which the polypeptide or polynucleotide ispresent in the cells or in the culture medium. For example, compositionsfor oral administration can form solutions, suspensions, tablets, pills,capsules, sustained release formulations, oral rinses or powders.

The terms “pharmaceutical composition” and “formulation” are usedinterchangeably.

The term “treatment” refers to any administration or application ofremedies for disease in a mammal and includes inhibiting the disease,arresting its development, relieving the disease, for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function; or stimulating an inefficient process. The termincludes obtaining a desired pharmacologic and/or physiologic effect,covering any treatment of a pathological condition or disorder in amammal. The effect may be prophylactic in terms of completely orpartially preventing a disorder or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse affect attributable to the disorder. It includes (1) preventingthe disorder from occurring or recurring in a subject who may bepredisposed to the disorder but is not yet symptomatic, (2) inhibitingthe disorder, such as arresting its development, (3) stopping orterminating the disorder or at least its associated symptoms, so thatthe host no longer suffers from the disorder or its symptoms, such ascausing regression of the disorder or its symptoms, for example, byrestoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, such as inflammation, pain and/or tumorsize.

The term “disease” refers to any condition, infection, disorder orsyndrome that requires medical intervention or for which medicalintervention is desirable. Such medical intervention can includetreatment, diagnosis and/or prevention.

The term “therapeutically effective amount” refers to an amount which,when administered to a living subject, achieves a desired effect on theliving subject. For example, an effective amount of the polypeptide ofthe invention for administration to the living subject is an amount thatprevents and/or treats an integrin αvβ3-mediated disease. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for systemic versus localized delivery,age, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art.

The term “T₁” refers to a point in time at which an etanerceptformulation has been stored for about one week at 40° C.

The term “T₂” refers to a point in time at which an etanerceptformulation has been stored for about two weeks at 40° C.

The term “T₄” refers to a point in time at which an etanerceptformulation has been stored for about four weeks at 40° C.

The term “M₃” refers, collectively, to three points in time, and inparticular to an analytical result being observed for an etanerceptformulation after duration of either about one, about two or about threemonths of storage at a storage temperature of 5° C. For example,reference herein to an analysis being conducted at M₃ should beunderstand to mean that such analysis is be done at the point in time atwhich etanercept formulation has been in storage for a duration selectedfrom about one, about two, or about three months. Thus, a requirementherein that an etanercept formulation elicit a certain analytical valueor measurement at M₃ is satisfied if the required value is observed at apoint in time corresponding to at least one of the following storagedurations: at approximately one month, at approximately two months or atapproximately three months of storage at 5° C.

The terms “Peak 1,” Peak 2″ and “Peak 3” when used herein in connectionwith discussion of HIC chromatography results refers to the same peaks1, 2 and 3 discussed in U.S. Pat. No. 7,294,481.

EMBODIMENTS OF THE INVENTION

When pharmaceutical compositions containing etanercept (Enbrel®),including aqueous and lyophilized formulations of etanercept are storedon a long term basis, the activity of etanercept can be lost ordecreased due to instability of the etanercept monomer via aggregationand/or chemical degradation including formation of fragments. Thus, thepresent invention provides several embodiments of aqueous formulationsof etanercept that allow stable long-term storage of etanercept, so thatetanercept is stable over the course of storage either in liquid orfrozen states. The provided formulations include, but are not limited toformulations that do not contain arginine and do not require any extrasteps such as rehydrating.

These embodiments are explained in a greater detail below.

Etanercept

All of the compositions of the present invention comprise etanercept(Enbrel®). As explained in the Background section of this application,etanercept is a dimeric fusion polypeptide consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)tumor necrosis factor receptor (TNFR) linked to the Fc portion of humanIgG1. Etanercept consists of 934 amino acids. The Fc component ofetanercept contains the constant heavy 2 (CH2) domain, the constantheavy 3 (CH3) domain and hinge region of human IgG1. An Fc domain cancontain one or all of the domains described above.

Etanercept suitable for storage in the present pharmaceuticalcomposition can be produced by living host cells that expressetanercept, such as hybridomas in the case of antibodies, or host cellsthat that have been genetically engineered to produce the polypeptide inthe case of fusion polypeptides or antibodies. Methods of geneticallyengineering cells to produce polypeptides are well known in the art.See, e.g., Ausubel et al., eds. (1990), Current Protocols in MolecularBiology (Wiley, New York). Such methods include introducing nucleicacids that encode and allow expression of the polypeptide into livinghost cells. These host cells can be bacterial cells, fungal cells, or,preferably, animal cells grown in culture. Bacterial host cells include,but are not limited to, Escherichia coli cells. Examples of suitable E.coli strains include: HB101, DH5.alpha, GM2929, JM109, KW251, NM538,NM539, and any E. coli strain that fails to cleave foreign DNA. Fungalhost cells that can be used include, but are not limited to,Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells. A fewexamples of animal cell lines that can be used are CHO, VERO, BHK, HeLa,Cos, MDCK, 293, 3T3, and W138. New animal cell lines can be establishedusing methods well know by those skilled in the art (e.g., bytransformation, viral infection, and/or selection). Optionally,etanercept can be secreted by the host cells into the medium.

Purification of the expressed etanercept can be performed by anystandard method. When etanercept is produced intracellularly, theparticulate debris is removed, for example, by centrifugation orultrafiltration. When etanercept is secreted into the medium,supernatants from such expression systems can be first concentratedusing standard polypeptide concentration filters. Protease inhibitorscan also be added to inhibit proteolysis and antibiotics can be includedto prevent the growth of microorganisms.

Etanercept can be purified using, for example, hydroxyapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, and any combination of known or yet to be discoveredpurification techniques, including but not limited to Protein Achromatography, fractionation on an ion-exchange column, ethanolprecipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSET®, an anion or cation exchange resinchromatography (such as a polyaspartic acid column), chromatofocusing,SDS-PAGE, and ammonium sulfate precipitation.

Etanercept Stabilized with Serine, Proline or Glutamate

The invention provides a stable aqueous pharmaceutical compositioncomprising etanercept and a stabilizer to inhibit instability,aggregation misfolding and/or fragmentation of the etanercept, whereinthe stabilizer comprises a compound selected from the group consistingof serine, proline and glutamate. In a preferred embodiment, thestabilizer comprises glutamate.

Without intending to be bound to any particular theory of the invention,it is believed that serine, proline and glutamate act as conformationalstabilizers to reduce etanercept's tendency to aggregate. The reductionin aggregation is believed to last for a long period of time, e.g., twoyears or more. It is believed that serine, proline and glutamate areable to stabilize aqueous pharmaceutical compositions containingetanercept because they are excluded from the surface of the protein,resulting in net conformation stabilization. The stabilizing effects ofserine, proline and/or glutamate include but are not limited to thebenefits of reduced aggregation of the etanercept monomer informulations containing the monomer.

The pharmaceutical compositions of the invention may be prepared bycombining a purified etanercept and a stabilizer. Further, a buffer, atonicity modifier and an additional excipient and other commonly usedinactive ingredients can be added as needed. For simplicity, these arediscussed more fully later in the specification. A person of ordinaryskill in the art will understand that the combining of the variouscomponents to be included in the composition can be done in anyappropriate order. For example, the buffer can be added first, middle orlast, and the tonicity modifier can also be added first, middle or last.A person of ordinary skill in the art will also understand that some ofthese chemicals can be incompatible in certain combinations, andaccordingly, are easily substituted with different chemicals that havesimilar properties but are compatible in the relevant mixture.

In a preferred embodiment, the concentration of the serine, proline orglutamate stabilizer in the provided formulations is preferably up toabout 150 mM.

Serine, proline and glutamate are available from commercial suppliers.

In an embodiment in which the stabilizer comprises glutamate, aformulation of the invention can comprise about 25 to about 50 mg/ml ofetanercept; up to 150 mM glutamate; less than about 6 wt. % sucrose;optionally up to about 100 mM NaCl; about 1 to about 30 mM sodiumphosphate, and wherein the formulation has pH 6.0 to about pH 7.0 andmore preferably about 6.0 to about 6.6, and most preferably betweenabout 6.3 to about 6.5.

In an embodiment in which the stabilizer comprises serine, a formulationof the invention can comprise about 25 to about 50 mg/ml of etanercept;less than about 150 mM serine; about 0.5 to about 3 wt. % sucrose; about1 to about 30 mM sodium phosphate, and wherein the formulation has pH6.0 to about pH 7.0 and more preferably about 6.0 to about 6.6, and mostpreferably between about 6.3 to about 6.5.

In an embodiment in which the stabilizer comprises proline, aformulation of the invention can comprise about 25 to about 50 mg/ml ofetanercept; less than about 150 mM proline; about 0.5 to about 3 wt. %sucrose; about 1 to about 30 mM sodium phosphate, about 15 to about 100mM NaCl; and wherein the formulation has pH 6.0 to about pH 7.0 and morepreferably about 6.0 to about 6.6, and most preferably between about 6.3to about 6.5.

Etanercept formulations according to the present invention comprisingserine, proline or glutamate are preferably characterized by an SECanalysis at T₂ of: about 80 to about 95 wt. % monomer content; less thanabout 4 wt. % aggregate(s) content; and less than about 8 wt. % fragment3 content.

In certain formulations of the invention containing serine, proline orglutamate for stabilization, the formulations elicit stability ascharacterized by:

-   -   (a) an SEC analysis at T₄ of greater than about 90, 91, 92, 93,        94, 95, 96, or 97 wt. % monomer content; and less than about 3,        2 or 1 wt. % aggregate(s) content; and    -   (b) an HIC analysis at T₂ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt. %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt. %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than about 20, 19, 18,        17, 16, 15, 14, or 13 wt. %; and    -   (c) an HIC analysis at T₄ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt. %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt. %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16,        15, 14 or 13 wt. %.

The terms “SEC”, “T₂” “T₄” “M₃” “HIC” “monomer content” “aggregate(s)”and “fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below.

In particularly preferred formulations containing serine, proline orglutamate for stabilization preferably are characterized by having anHIC analysis at T₄ or T₂ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 1%; theamount of the composition represented by peak 2 of the HIC chromatogramis greater than about 95 wt. %, and most preferably greater than about99 wt. %; and the amount of the composition represented by peak 3 of theHIC chromatogram is less than about 3 wt. %.

A further preferred formulation using serine, proline and/or glutamatefor stabilization of etanercept comprises about 50 mg/ml etanercept;less than about 150 mM serine, proline or glutamate, and most preferablyglutamate; about 0 to 3% sucrose; about 1 to 30 mM phosphate buffer, andhaving a pH of about 6.0 to 6.6; and characterized by: an SEC analysisat T₄ of greater than about 97 wt. % monomer content and less than about1 wt. % aggregate(s) content; an HIC analysis at T₂ wherein the amountof the composition represented by peak 1 of the HIC chromatogram is lessthan about 3 wt. %; the amount of the composition represented by peak 2of the HIC chromatogram is greater than about 82 wt. %; and the amountof the composition represented by peak 3 of the HIC chromatogram is lessthan about 15 wt. %; and an HIC analysis at T₄ wherein the amount of thecomposition represented by peak 1 of the HIC chromatogram is less thanabout 2 wt. %; the amount of the composition represented by peak 2 ofthe HIC chromatogram is greater than about 84 wt. %; and the amount ofthe composition represented by peak 3 of the HIC chromatogram is lessthan about 13 wt. %.

In another embodiment of the invention, the stabilized etanerceptcomposition is stabilized with serine, proline or glutamate, theformulation is free or essentially free of arginine, and the formulationelicits long term storage stability as characterized by at least one of:

-   -   SEC analysis at M₃ of: monomer content greater than about 90%;        aggregates content of less than about 3 wt %; and fragment 3        content less than about 5 wt %: and    -   HIC analysis at M₃ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3 wt. %; the amount of the composition represented by peak 2 of        the HIC chromatogram is greater than 80 wt. %; and the amount of        the composition represented by peak 3 of the HIC chromatogram is        less than about 20 wt. %.

Preferably the serine, proline or glutamate stabilized composition theinvention elicits long term storage stability as characterized by: anHIC analysis at M₃ wherein the amount of the composition represented bypeak 2 of the HIC chromatogram is greater than or equal to about 95 wt.%; and wherein, if peak 3 is present on the HIC chromatogram, the amountof the composition represented by peak 3 is less than or equal to about1 wt. %.

In a further aspect of the invention the serine, proline or glutamatestabilized composition of the invention elicits long term storagestability as characterized by:

-   -   SEC analysis at M₃ or T₄ of greater than about 90 wt. % monomer        content; less than about 3 wt. % aggregate(s) content; and less        than about 5 wt % fragment 3; and    -   HIC analysis at M₃ or T₂ or T₄ wherein the amount of the        composition represented by peak 1 of the HIC chromatogram is        less than about 3 wt. %; the amount of the composition        represented by peak 2 of the HIC chromatogram is greater than 80        wt. %; and the amount of the composition represented by peak 3        of the HIC chromatogram is less than about 20 wt. %.

A further preferred glutamate stabilized etanercept formulationcomprises: about 50 mg/ml etanercept; about 120 mM glutamate; about 1%sucrose, and about 25 mM phosphate; having a pH of about 6.3 to about6.5, and exhibiting the SEC and HIC analytical characteristicsreferenced above.

Preferably, a stabilized etanercept formulation according to theinvention after storage at 5° C. or 25° C. for one, two or three monthsis (a) characterized by an HIC chromatogram in which peak 3 isessentially absent; (b) characterized by an SEC chromatogram whichcontains no, or essentially no peak corresponding to aggregate(s); and(c) characterized by an SEC chromatogram in which the monomer contentrepresents at least about 97 wt % of the composition.

The serine, proline or glutamate stabilized etanercept formulations,containing no arginine, are found to elicit stability over three monthsstorage at 5 C that is comparable to or better than the presentlyavailable arginine containing formulation of commercially availableEnbrel. Accordingly the present invention is further directed to serine,proline or glutamate stabilized formulations of etanercept, whichcontain no arginine, or are essentially free of arginine, and whereinthe composition, at M₃ or T₂ or T₄, elicits long term storage stabilitythat meets one or both of the following criteria:

-   -   (A) stability comparable to or better than commercially        available etanercept marketed under the trademark Enbrel®, as        measured by (i) SEC analysis of the amounts of aggregate(s),        monomer and fragment 3 in the composition (as defined in the        specification) and (ii) HIC analysis of amounts of material in        the composition corresponding to peaks 1, 2 and 3 of the HIC        chromatogram (as defined in the specification); and    -   (B) an HIC chromatogram in which (i) peak 3 is absent, or        essentially absent and (ii) peak 2 represents greater than about        95 wt % of the composition; an SEC chromatogram containing        essentially no peak corresponding to aggregate(s); and an SEC        chromatogram in which the monomer content represents at least        about 95 wt % of the composition.

Although the invention does not exclude the use of arginine, theetanercept formulations comprising serine, proline and/or glutamate forstabilization according to the present invention do not require argininefor stabilization, and are therefore preferably free or essentially freeof arginine.

Additional Components of the Provided Pharmaceutical Compositions

The formulations of the invention may also include buffers, tonicitymodifiers, excipients, pharmaceutically acceptable carriers and othercommonly used inactive ingredients of the pharmaceutical compositions.For simplicity, these are discussed more fully later in the application.

Buffers maintain pH in a desired range. Suitable buffers includehistidine, potassium phosphate, sodium or potassium citrate, maleicacid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris),various forms of acetate and diethanolamine. The concentration of thebuffer in the formulation is preferably between about 1 mM to about 1M,and more preferably about 10 mM to about 200 mM. Buffers are well knownin the art and are manufactured by known methods and available fromcommercial suppliers.

Examples of suitable buffers are phosphate, histidine, citrate, maleate,tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris),bicarbonate.

In a preferred embodiment, the buffer is sodium phosphate.

In a preferred embodiment, the pH of the pharmaceutical composition isat or near physiological levels. Thus, preferably, the pH of theprovided compositions is between about 5.8 and about 8.4; and even morepreferably, between about 6.2 and about 7.4. A person of ordinary skillin the art will understand that the pH can be adjusted as necessary tomaximize stability and solubility of etanercept in a particularformulation. Thus, etanercept formulations at a pH outside ofphysiological ranges, yet tolerable to the patient, are also within thescope of the invention.

A tonicity modifier is a molecule that contributes to the osmolality ofa solution. The osmolality of a pharmaceutical composition is preferablyadjusted to maximize the active ingredient's stability and/or tominimize discomfort to the patient upon administration. It is generallypreferred that a pharmaceutical composition be isotonic with serum,i.e., having the same or similar osmolality, which is achieved byaddition of a tonicity modifier.

In a preferred embodiment, the osmolality of the provided formulationsis from about 180 to about 420 mOsM. However, it is to be understoodthat the osmolality can be either higher or lower as specific conditionsrequire.

Examples of tonicity modifiers suitable for modifying osmolalityinclude, but are not limited to amino acids (not including arginine)(e.g., cysteine, histidine and glycine), salts (e.g., sodium chloride,potassium chloride and sodium citrate) and/or saccharides (e.g.,sucrose, glucose and mannitol).

Preferred tonicity modifiers are glycine, alanine, sodium chloride,potassium chloride, and sodium sulfate.

In a preferred embodiment, the concentration of the tonicity modifier inthe formulation is preferably between about 1 mM to about 1 M, morepreferably about 10 mM to about 200 mM. Tonicity modifiers are wellknown in the art and are manufactured by known methods and availablefrom commercial suppliers.

Excipients, also referred to as chemical additives, co-solutes, orco-solvents, that stabilize the polypeptide while in solution (also indried or frozen forms) can also be added to a pharmaceuticalcomposition. Excipients are well known in the art and are manufacturedby known methods and available from commercial suppliers.

Examples of suitable excipients include but are not limited tosugars/polyols such as: sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose; polymers such as: serumalbumin (bovine serum albumin (BSA), human SA or recombinant HA),dextran, poly(viny alcohol) PVA, hydroxypropyl methylcellulose (HPMC),polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP),hydroxyethylcellulose (HEC); non-aqueous solvents such as: polyhydricalcohols, (e.g., PEG, and glycerol) and dimethylformamide (DMF); aminoacids such as: proline, L-serine, sodium glutamic acid, alanine,glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric acid;surfactants such as: Tween®-80 (polysorbate 80), Tween®-20 (polysorbate20), SDS, polysorbate, poloxamers; and miscellaneous excipients such as:potassium phosphate, sodium acetate, ammonium sulfate, magnesiumsulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions(e.g., zinc, calcium, and magnesium), CHAPS, monolaurate,2-O-beta-mannoglycerate or any combination of the above.

Preferred excipients are sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose, bovine serum albumin(BSA), human serum albumin (HSA), recombinant albumin, dextran, PVA,hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), polyethyleneglycol, ethylene glycol, glycerol, alanine, glycine, lysinehydrochloride, sarcosine, SDS, polysorbate 20, polysorbate 80, poloxamer188, trimethylamine N-oxide, betaine, zinc ions, calcium ions, magnesiumions, CHAPS, sucrose monolaurate, and 2-O-beta-mannoglycerate.

The concentration of one or more excipients in a formulation of theinvention is/are preferably between about 0.001 to 5 weight percent,more preferably about 0.1 to 2 weight percent.

Methods of Treatment

In another embodiment, the invention provides a method of treating amammal comprising administering a therapeutically effective amount ofthe pharmaceutical compositions of the invention to a mammal, whereinthe mammal has a disease or disorder that can be beneficially treatedwith etanercept.

In a preferred embodiment, the etanercept is derived from the samespecies of mammal as is to be treated with the composition.

In a preferred embodiment, the mammal is a human.

Diseases or disorders that can be treated with the provided compositionsinclude but are not limited to rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Wegener's disease (granulomatosis),Crohn's disease (or inflammatory bowel disease), chronic obstructivepulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia,psoriasis, and atopic dermatitis. Additional diseases or disorders thatcan be treated with the compositions of the present invention includethose described in WO 00/62790, WO 01/62272, U.S. Patent Application No.2001/0021380, and U.S. Pat. No. 7,648,702 B2, the relevant portions ofwhich are incorporated herein by reference.

The provided pharmaceutical compositions may be administered to asubject in need of treatment by injection systemically, such as byintravenous injection; or by injection or application to the relevantsite, such as by direct injection, or direct application to the sitewhen the site is exposed in surgery; or by topical application.

In one embodiment, the invention provides a method of treatment and/orprevention of rheumatoid arthritis comprises administering to a mammalin need thereof a therapeutically effective amount of one of theprovided etanercept compositions.

The therapeutically effective amount of the etanercept in the providedcompositions will depend on the condition to be treated, the severity ofthe condition, prior therapy, and the patient's clinical history andresponse to the therapeutic agent. The proper dose can be adjustedaccording to the judgment of the attending physician such that it can beadministered to the patient one time or over a series ofadministrations.

In one embodiment, the effective etanercept amount per adult dose isfrom about 1-500 mg/m², or from about 1-200 mg/m², or from about 1-40mg/m² or about 5-25 mg/m².

Alternatively, a flat dose may be administered, whose amount may rangefrom 2-500 mg/dose, 2-100 mg/dose or from about 10-80 mg/dose.

If the dose is to be administered more than one time per week, anexemplary dose range is the same as the foregoing described dose rangesor lower and preferably administered two or more times per week at a perdose range of 25-100 mg/dose.

In another embodiment, an acceptable dose for administration byinjection contains 80-100 mg/dose, or alternatively, containing 80 mgper dose.

The dose can be administered weekly, biweekly, or separated by severalweeks (for example 2 to 8).

In one embodiment, etanercept is administered at 25 to 75 mg/ml by asingle subcutaneous (SC) injection.

In some instances, an improvement in a patient's condition will beobtained by administering a dose of up to about 100 mg of thepharmaceutical composition one to three times per week over a period ofat least three weeks. Treatment for longer periods may be necessary toinduce the desired degree of improvement. For incurable chronicconditions the regimen may be continued indefinitely. For pediatricpatients (ages 4-17), a suitable regimen may involve administering adose of 0.4 mg/kg to 5 mg/kg of etanercept, one or more times per week.

In another embodiment, the pharmaceutical formulations of the inventionmay be prepared in a bulk formulation, and as such, the components ofthe pharmaceutical composition are adjusted to be higher than would berequired for administration and diluted appropriately prior toadministration.

The pharmaceutical compositions can be administered as a soletherapeutic or in combination with additional therapies as needed. Thus,in one embodiment, the provided methods of treatment and/or preventionare used in combination with administering a therapeutically effectiveamount of another active agent. The other active agent may beadministered before, during, or after administering the pharmaceuticalcompositions of the present invention. Another active agent may beadministered either as a part of the provided compositions, oralternatively, as a separate formulation.

Administration of the provided pharmaceutical compositions can beachieved in various ways, including parenteral, peroral, buccal,sublingual, nasal, rectal, intraperitoneal, intradermal, transdermal,subcutaneous, intravenous, intra-arterial, intracardiac,intraventricular, intracranial, intratracheal, intrathecaladministration, intramuscular injection, intravitreal injection, andtopical application.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, intraperitoneal, intracerebrospinal,intra-articular, intrasynovial, intravitreal, and/or intrathecal.Parenteral administration can be by bolus injection or continuousinfusion. Pharmaceutical compositions for injection may be presented inunit dosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In addition, a number of recent drug deliveryapproaches have been developed and the pharmaceutical compositions ofthe present invention are suitable for administration using these newmethods, e.g., Inject-ease®, Genject®, injector pens such as GenPen®,and needleless devices such as MediJector® and BioJector®. The presentpharmaceutical composition can also be adapted for yet to be discoveredadministration methods. See also Langer, 1990, Science, 249:1527-1533.

The provided pharmaceutical compositions can also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions may, if desired, be presented in a vial,pack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. In one embodiment the dispenser devicecan comprise a syringe having a single dose of the liquid formulationready for injection. The syringe can be accompanied by instructions foradministration.

In another embodiment, the present invention is directed to a kit orcontainer, which contains an aqueous pharmaceutical composition of theinvention. The concentration of the polypeptide in the aqueouspharmaceutical composition can vary over a wide range, but is generallywithin the range of from about 0.05 to about 20,000 micrograms permilliliter (μg/ml) of aqueous formulation. The kit can also beaccompanied by instructions for use.

The present invention is more particularly described in the followingexamples that are intended as illustrative only, since manymodifications and variations therein will be apparent to those skilledin the art.

Example 1 Etanercept Stabilized with Serine

Etanercept formulations with serine, proline or glutamate stabilizationmay be prepared using the following procedures:

Each solid formulation component is weighed to the amount required for agiven volume of formulation buffer. These components are combined into abeaker or vessel capable of carrying and measuring the given volume offormulation buffer. A volume of deionized water equal to approximately ¾of the target given formulation buffer is added to the beaker, and thecomponents are then solublized. The pH of the buffer is adjusted to thetarget formulation pH using 1 M sodium hydroxide and/or 1 M hydrogenchloride. The final formulation buffer volume is then raised to thetarget volume through the addition of deionized water. Etanerceptprotein solution is placed in dialysis material housing (such as ThermoScientific Slide-A-Lyzer MINI Dialysis Unit 10,000 MWCO), which is thenplaced in contact with the desired formulation buffer for 12 hours at 4°C. Formulation buffer volume to protein solution volume ratio should beno less than 1000:1. The dialysis housing and protein solution itcontains is then placed in a second, equal volume of formulation bufferfor an additional 12 hours at 4° C. Resulting protein solution isremoved from the dialysis material housing, and the concentration ofprotein determined using ultraviolet spectroscopy. Protein concentrationis adjusted to the desired level using centrifugation (such as AmiconUltra 10,000 MWCO Centrifugal Concentrators) and/or dilution withformulation buffer.

The compositions can be tested for long-term stability by size exclusionchromatography (SEC), denatured SEC (dSEC), hydrophobic interactionchromatography (HIC), sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE), and for binding and bioactivity at varioustimepoints. The bioactivity can be measured by any number of well-knownassays.

For example, the techniques of Size Exclusion Chromatography aredescribed in Hawe et al, Pharm. Res. 2011, 28: 2302 and/or vanMarrschalkerweerd et al., Eur. J. Pharm. Biopharm. 2011, 78: 213.Similarly, the techniques of Denatured Size Exclusion Chromatography,Hydrophobic Interaction Chromatography, and SodiumDodecylSulfate-PolyAcrylamide Gel Electrophoresis are also well known topersons having ordinary skill in the art.

It is believed that the composition will be stable over the term of twoyears or more.

Formulation 1:15

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 25 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 1% (w/v) NaCl (inactive) 100 mM

Formulation 1:12

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 25 mM Sodium phosphate, pH 6.4 (inactive) 25 mMSucrose (inactive) 2.5% (w/v) or 5% (w/v) NaCl (inactive) 100 mM

Formulation 1:16

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 50 mM Sodium phosphate, pH 6.4 (inactive) 25 mMSucrose (inactive) 5% (w/v) NaCl (inactive) 25 mM

Formulation 2:4

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 100 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 1% (w/v)

Formulation 3:8

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Serine(inactive ingredient) 120 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 1% (w/v)

Example 2 Etanercept Stabilized with Proline Formulation 1:4

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Proline(inactive ingredient) 25 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 2.5% (w/v) NaCl (inactive) 50 mM

Formulation 1:5

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Proline(inactive ingredient) 50 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 1.0% (w/v) NaCl (inactive) 25 mM

Formulation 1:6

Ingredient concentration Etanercept (active ingredient) 50 mg/ml Proline(inactive ingredient) 100 mM Sodium phosphate, pH 6.3 (inactive) 25 mMSucrose (inactive) 1.0% (w/v)

Example 3 Etanercept Stabilized with Glutamate

Compositions stabilized with glutamate may be prepared and tested usingthe procedures similar to those described in Example 1.

Glutamate stabilized etanercept compositions, containing no arginine,are exemplified below:

Formulation 1:9

Ingredient concentration Etanercept (active ingredient) 50 mg/mlGlutamate (inactive ingredient) 25 mM Sodium phosphate, pH 6.3(inactive) 25 mM Sucrose (inactive) 1% (w/v) NaCl (inactive) 100 mM

Formulation 2:2

Ingredient concentration Etanercept (active ingredient) 50 mg/mlGlutamate (inactive ingredient) 50 mM Sodium phosphate, pH 6.3(inactive) 25 mM Sucrose (inactive) 1% (w/v) NaCl (inactive) 50 mM

Formulation 2:3

Ingredient concentration Etanercept (active ingredient) 50 mg/mlGlutamate (inactive ingredient) 100 mM Sodium phosphate, pH 6.3(inactive) 25 mM Sucrose (inactive) 1% (w/v)

Formulation 3:5

Ingredient concentration Etanercept (active ingredient) 50 mg/mlGlutamate (inactive ingredient) 120 mM Sodium phosphate, pH 6.5(inactive) 25 mM Sucrose (inactive) 1% (w/v)The composition can be tested for long-term stability, and thebioactivity can be measured in the same fashion as discussed in Example1.

It is believed that the composition will be stable over the term of twoyears or more.

Example 4 Preparation of Etanercept

Step 1. Cell Expansion.

In a manner known in the art, cell expansion necessary to generate asufficient number of cells for inoculation of a production bioreactor isperformed using a clone of CHO cells expressing the etanercept fusionprotein. The product of this expression process (a harvested cellculture fluid) results in a mixture of correctly folded etanercept, aswell as incorrectly folded and/or aggregated etanercept, along withadditional impurities. The harvested cell culture fluid comprising suchprotein mixture is subjected to detergent viral inactivation.

Step 2.

Affinity Chromatography. Affinity chromatography is performed on theharvested cell culture obtain in Step 1 above using a conventionalProtein A affinity column in a well known manner. Product recovery isapproximately 85%. The product obtained is a complex protein mixturecomprising correctly folded etanercept, incorrectly folded etanercept,and/or aggregates of correctly and/or incorrectly folded etanercept, orprotein fragments. The product obtained from this Protein A affinitycolumn purification step is adjusted to pH 3.5 and then subjected to aviral inactivation step. Following viral inactivation the product isadjusted to pH 5.5 and then further clarified in a known manner using acommercially obtained capsule filter.

Step 3A.

Mixed-Mode Cation Exchange Chromatography. A 31.8 L (45 cm diameter×20cm bed height) packed bed GE Healthcare Capto MMC chromatography columnis used to purify the product obtained in Step 2 above. Prior to use,the column is equilibrated with 2 CV of 25 mM acetate pH 5.5 andsanitized with 2 CV of 0.1 N NaOH, 1 M NaCl and neutralized with 2 CV of25 mM acetate, 0.7 M NaCl, pH 5.5. The column is then equilibrated with8-10 CV of 25 mM acetate pH 5.5 until the effluent is pH 5.5 and 3.5mS/cm. The Protein A pool from step 2 above is diluted to ≦6 mS/cm withWFI and applied to a column loading of up to 15 g/L media for eachcycle. The column is operated at a linear velocity of 200 cm/h to give a6 minute residence time. After loading, the column is washed with 2 CVof 25 mM acetate pH 5.5. The product is then eluted with an 8.5 CV, 15%to 85% gradient of 25 mM acetate pH 5.5 to 25 mM acetate, 0.7 M NaCl, pH5.5. Product collection begins at 0.15 OD (A280, 1.0 cm path length) andcollection ends at 50% of peak maximum. The eluate volume isapproximately 5 CV. Residual product and contaminants are stripped fromthe column with 2 CV of 10 mM Tris, 1 M NaCl, pH 8.0 and discarded. Theproduct obtained from the mixed mode column is filtered using aMillipore Opticap XL10, 0.22 μm Durapore capsule filter, (0.69 m²). Theproduct obtained from this step represents a recovery of about 70% ofthe Protein A material obtained in Step 2

Step 3B.

Mixed Mode Anion Exchange Chromatopgraphy. A 27.0 L (45 cm diameter×17cm bed height) packed bed GE Healthcare Capto Adhere chromatographycolumn is used to further purify the product obtained in step 3A above.Prior to use, the column is equilibrated with 2 CV of 25 mM Tris, pH 8.0and sanitized with 2 CV of 0.1 N NaOH, 1M NaCl and neutralized andequilibrated with 2 CV of 25 mM Tris, pH 8.0. Prior to product loading,the column is equilibrated with 3 CV of 10 mM Tris, pH 8.0. The CaptoMMC pool from Step 3A above is adjusted to pH 8.1 with ˜0.045 kg of 1 MTris, pH 8.3 per kg of pool. The product from Step 3A above was dilutedin-line 1:3.8 with WFI to adjust the conductivity to 12.0 mS/cm and pH8.0. The resulting material is then applied to a column loading of up to15 g/L media. The column is operated at a linear velocity of 170 cm/h togive a 6 minute residence time. After loading, the column is washed with2 CV of 25 mM Tris, pH 8.0. The product is then eluted with a 10 CVgradient (20% to 90%) of 25 mM Tris, pH 8.0 to 10 mM Tris, 1 M NaCl, pH8.0. Product collection is started at 0.15 OD (A280, 1.0 cm path length)and collection ended at 25% of peak maximum. The eluate volume is 4-6CV. The eluted product is filtered using a commercially availablecapsule filter and then subjected in a known manner to viral filtrationand tangential flow filtration steps. Overall product recovery from step3B (including the final viral and tangential flow filtration steps) wasapproximately 68%. Product recovery measured before the filtration stepswas about 75%. A schematic representation of HIC data obtained oneluation fractions from this step are representing in FIG. 12.

Analysis:

The final filtered product obtained in this example is found to havegreater than about 90 wt % correctly folded etanercept as determined byHIC; less than 5 wt % incorrectly folded etanercept species asdetermined by HIC; less than about 3 wt % of clipped material by HICanalysis (believed to be fragments of etanercept in which the TNFRportion thereof has been truncated) and a combined amount of correctlyand incorrectly folded etanercept of greater than 95 wt % as determinedby size exclusion chromatography.

Analysis of Etanercept Formulations

A. Thermal Stability Storage

Following dialysis and concentration, samples of the etanerceptformulations exemplified above were sterile filtered in a bio safetycabinet. Using sterilized pipettes and autoclaved pipette tips, samplesof the etanercept formulations were transferred to pre-labeled andautoclaved 1 mL lyophilization vials. Vials were stoppered with sterilebutyl stoppers and crimped with aluminum caps. All vials were thentransferred to thermal stability ovens. Samples were subject to twothermal stability regimes: (1) two weeks at 40° C. and (2) four weeks at25° C. Throughout this specification, these two temperature regimes aredenoted “T₂” and T₄,” respectively.

B. Size Exclusion Chromatography (SEC)

Etanercept formulations disclosed herein were analyzed using the wellknown technique of Size Exclusion Chromatography (SEC), ahigh-performance liquid chromatography method in which analytes areseparated by size (see Rogner, M. (2000). Size Exclusion Chromatography.Protein Liquid Chromatography. M. Kastner. Amsterdam, Elsevier. 61:89-145.). In order to evaluate thermal stability of the Etanerceptsamples described above, the samples were examined by a SEC method basedon the literature (van Maarschalkerweerd, A., G. J. Wolbink, et al.(2011). “Comparison of analytical methods to detect instability ofetanercept during thermal stress testing.” European Journal ofPharmaceutics and Biopharmaceutics 78(2): 213-221.) The mobile phasebuffer was prepared to contain 50 mM sodium phosphate monobasicmonohydrate and 150 mM arginine. The pH was adjusted to 6.5 using 1 MHCl. All separations were performed using a Tosoh TSK-Gel SWxl 6 mm×4 cmguard column (cat. no. 8543) attached linearly to a Tosoh TSK-Gel G4000SWxl 7.8 mm×30 cm (cat. no. 8542). To perform a separation, the columnswere brought to room temperature (23° C.) and equilibrated with mobilephase at a flow rate of 0.5 mL/min. 5 microliters of 50 mg/mL etanerceptformulation were injected onto the column using an autosampler. Theseparation was accomplished over 30 minutes at a flow rate of 0.5mL/minute. Column eluent was monitored at a wavelength of 280 nm duringthis time.

C. Integration of Size Exclusion Chromatography Chromatograms

All integration was performed using Chromeleon software (Dionex). Priorto integration, the SEC chromatogram for a buffer containing noetanercept was subtracted from all chromatograms. All integration wasperformed between retention times of 12 minutes and 26 minutes. Severalparameters were used to define a peak. The minimum area for a detectedpeak was set to 0.05 mAu*min. The two-dimensional sensitivity for peakdetection was set to 0.01 mAu and 75 seconds. Peak shoulders were addedmanually using a manual integration tool. All detected peaks weremanually adjusted in two steps. First, peak baselines (the bottomboundary of the peak) were adjusted to horizontal. Secondly, thevertical positions of the peak baselines were adjusted to that of thechromatogram baseline. The chromatogram baseline value was defined asthe signal in absence of analyte. The signal in absence of analyte wasdefined as the absorbance in mAu at 12 minutes retention time.

D. SEC Fractions of Etanercept Formulations

In the SEC analysis of etanercept formulations described above, threeSEC chromatography fractions were identified and studied. The fractionsthat were analyzed were, in the order of elution from the SEC column:(1) a high molecular weight fraction representing aggregates of theintact etanercept TNFR:FC fusion protein likely assembled vianon-covalent electrostatic attraction among intact etanercept molecules(hereinafter “aggregate(s)” or aggregate(s) content); (2) monomercontent, representing the intact etanercept TNFR:Fc fusion protein(hereinafter referred to as “monomer” of “monomer content”); (3) afraction likely representing one fragment or a population of fragmentsof the etanercept molecule in which one portion of the TNFR:moleculefusion protein has become cleaved from the monomer; in the loss of anarm of the Fabportion of the fusion protein at the hinge region of themolecule. The most common fragment or clipped species, as measured bySEC, is referred to as Fragment 3. In conducting the SEC analysis, itwill be observed that aggregates elute first, followed by monomer,followed by fragment 3.

The following tables shows the relative amounts of Aggregates, Monomerand Fragment 3 determined by SEC analysis as described above.

TABLE I SEC ANALYSIS OF MONOMER Formulation No. t₀ t₁ t₂ CommercialEnbrel 98.81 92.58 87.64 (comparative) [1:2] 1:5 98.38 91.65 86.89 1:998.48 92.05 86.06 2:2 98.11 — 86.92 2:3 98.14 — 88.84 2:4 98.12 — 88.163:5 98.35 — 90.75 3:8 98.15 — 89.27 Note: Amounts reported Tables I, IIand III are percentages by weight t₀ = formulation maintained at 5 C.and analyzed within 24 hours of creation. t₁ = formulation stored forone week at 40° C. t₂ = formulation stored for two weeks at 40 C.

TABLE II SEC ANALYSIS OF AGGREGATES Formulation No. t₀ t₁ t₂ CommercialEnbrel 0.09 0.59 1.02 (comparative) 1:5 0.23 0.63 1.01 1:9 0.18 0.672.20 2:2 0.29 — 3.53 2:3 0.29 — 2.31 2:4 0.29 — 2.29 3:5 0.28 — 0.99 3:80.28 — 1.60 Note: Amounts reported Tables I, II and III are percentagesby weight t₀ = formulation maintained at 5 C. and analyzed within 24hours of creation. t₁ = formulation stored for one week at 40° C. t₂ =formulation stored for two weeks at 40 C.

TABLE III ANALYSIS OF FRAGMENT 3 Formulation No t₀ t₁ t₂ CommercialEnbrel 0.00 3.30 6.29 (comparative) 1:5 0.00 4.43 6.64 1:9 0.00 3.966.34 2:2 0.00 — 4.67 2:3 0.00 — 3.61 2:4 0.00 — 3.61 3:5 0.00 3.15 3:80.00 3.61 Note: Amounts reported Tables I, II and III are percentages byweight t₀ = formulation maintained at 5 C. and analyzed within 24 hoursof creation. t₁ = formulation stored for one week at 40° C. t₂ =formulation stored for two weeks at 40 C.

TABLE IV SEC ANALYSIS OF MONOMER CONTENT (T₄ = 4 weeks/25° C.)FORMULATION T₀ T₄ No. Monomer Content Monomer Content Commercial Enbrel98.15 97.86 (comparative) 3:5 98.35 95.16 3:8 98.15 97.65Table IV below shows monomer (etanercept) content of etanerceptformulations prepared according to the present invention, when storedfor four weeks at 25° C.-denoted by the symbol T₄. In the followingtable T₀ represents SEC measurements conducted within 24 hours offormulation preparation, at sample temperature of 5° C.; and T₄represents etanercept formulation samples subjected to SEC analysisafter 4 weeks storage at 25° C.

TABLE V SEC ANALYSIS OF AGGREGATES CONTENT (T₄ 4 weeks/25° C.)FORMULATION T₀ T₄ No. Aggregate(s) Content Aggregate(s) ContentCommercial Enbrel 0.28 0.25 (comparative) 3:5 — 0.50 3:8 0.28 0.37

Table V below shows aggregate(s) content of etanercept formulationsprepared according to the present invention after storage for four weeksat 25° C. In the following table T₀ represents SEC measurementsconducted within 24 hours of formulation preparation, at sampletemperature of 5° C.; and T₄ represents etanercept formulation samplessubjected to SEC analysis after 4 weeks storage at 25° C.

HIC Analysis of Etanercept Formulations

The following tables (Tables VI and VII) show the results of hydrophobicinteraction chromatography (“HIC chromatography”) conducted on samples3:5 and 3:8. HIC chromatography was carried out in a manner known in theart and generally described in U.S. Pat. No. 7,294,481, incorporatedherein by reference. Samples were evaluated at t₀ (within 24 hours ofpreparation at 5° C.) and again after either two weeks of storage at 25°C. (t₂) (see Table VI) or after 4 weeks of storage at 25° C. (t₄) (SeeTable VII) Peak 1 in the HIC chromatogram is believed to be or include“Fragment 3”, which is identified and quantified using SEC, asreferenced above in the discussion of SEC data; Peak 2 is etanerceptmonomer as referenced above in the discussion of SEC data; and Peak 3includes “Aggregate(s)” as referenced above in the discussion of SECdata. It should further be understood that the terms “peak 1”, “peak 2”and “peak 3 as used here also constitute a reference to the HIC peak 1,peak 2 and peak 3 referred to and disclosed in FIG. 4 of U.S. Pat. No.7,294,481 incorporated herein by reference.

TABLE VI HIC Data after Two Weeks Storage at 40° C. PEAK 1 PEAK 2 PEAK 3Form. # T₀ T₂ T₀ T₂ T₀ T₂ Enbrel 0.91 3.23 86.72 83.41 12.33 13.36(comparative) 3:5 0.72 2.95 85.82 82.50 13.45 14.55 3:8 0.72 3.08 85.8083.90 13.48 13.02

TABLE VII HIC Data after Storage at 25° C. for 4 Weeks (T₄) PEAK 1 PEAK2 PEAK 3 Form. # T₀ T₄ T₀ T₄ T₀ T₄ Commercial 0.91 1.09 86.76 86.9512.33 11.97 Enbrel (comparative) 3:5 0.54 1.10 85.12 84.06 14.33 14.843:8 0.72 1.20 85.80 85.98 13.48 12.82

Tables VIII through XVI, below, contain the results of stability testingconducted on formulation 3:5 (see Example 3 above) and containingetanercept material produced in the manner generally described Example 4(Preparation of Etanercept). Stability of this formulation 3:5 wasevaluated using SEC, HIC and FlowCam analysis for subvisible particles,based on one, two and three months of storage at various temperatures,including 5° C. Following is the methodology used to conduct thesestability experiments:

Bulk Etanercept Storage.

Non-formulated bulk etanercept was stored at 2-8° C. as indicated by theEnbrel® package insert.

UV Spectroscopy.

UV spectroscopy was used to determine protein concentration in variousstability samples. The absorbance at 280 nm of bulk substance (50 mg/mLEnbrel) was determined to be 0.625 using a 0.1 mm pathlength cell,leading to an extinction coefficient of 1.30 mL/mg*cm. This value wasused for all calculations in this project.

Dialysis and Concentration of Etanercept Formulation.

All buffers were prepared in two 1 L volumes containing all buffercomponents. Bulk material was loaded into Slide-A-Lyzer dialysis units(10 kD cutoff, one to three mL volume) following a five-minute rinse ofthe cassettes in deionized water. Dialysis samples were subjected to afive-hour dialysis at 2-8° C. in 1 L of buffer, followed by a seconddialysis at 2-8° C. overnight in a second 1 L of buffer. When dialyzingthe etanercept formulation 3:5, two 4 L dialysis events were used as 24mL of stock protein was needed for the formulation.

All samples were concentrated above their target value using AmiconUltra 10K cutoff centrifuge filters (2 mL size). UV was used todetermine new concentration of samples, which were then diluted to theappropriate level using formulation buffer.

Thermal Stability Sample Incubation.

Following dialysis and concentration, thermal stability samples weresterile filtered in a bio safety cabinet. Using sterilized pipettes andautoclaved pipette tips, samples were transferred to pre-labeled andautoclaved 1 mL lyophilization vials. Vials were stoppered with sterilebutyl stoppers and crimped with aluminum caps. All vials were thentransferred to thermal stability ovens.

Size Exclusion Chromatography (SEC).

Size exclusion chromatography (SEC) was performed using differentmethods. In one of the SEC methods identified herein as “Method 2” themobile phase buffer was prepared to contain 50 mM sodium phosphatemonobasic monohydrate and 150 mM arginine HCl. The pH was adjusted to6.5 using 1 M NaOH. Separations were performed using a Phenomonex Yarra3 micron SEC 3000, 30 cm×4.6 mm. To perform a separation, the columnswere brought to room temperature (23° C.) and equilibrated with mobilephase at a flow rate of 0.5 mL/min. One microliter of 50 mg/mLetanercept formulation were injected onto the column using anautosampler. The separation was accomplished over 10 minutes at a flowrate of 0.5 mL/minute. Column eluent was monitored at a wavelength of280 nm during this time.

In an alternate SEC method, hereinafter referred to as “Method 3” NaClwas used as a salt for the mobile phase at a concentration of 100 mM, pH6.3, in replacement of arginine HCl.

In a further alternate SEC method, referred to herein as Method 1, theSEC analysis was conducted as follows: The mobile phase buffer wasprepared to contain 50 mM sodium phosphate monobasic monohydrate and 150mM arginine. The pH was adjusted to 6.5 using 1 M HCl. All separationwere performed using a Tosoh TSK-Gel SWxl 6 mm×4 cm guard column (cat.no. 8543) attached linearly to a Tosoh TSK-Gel G4000 SWxl 7.8 mm×30 cm(cat. no. 8542). To perform a separation, the columns were brought toroom temperature (23° C.) and equilibrated with mobile phase at a flowrate of 0.5 mL/min. 5 microliters of 50 mg/mL etanercept formulationwere injected onto the column using an autosampler. The separation wasaccomplished over 30 minutes at a flow rate of 0.5 mL/minute. Columneluent was monitored at a wavelength of 280 nm during this time.

Integration of Size Exclusion Chromatography Chromatograms.

All integration was performed using Chromeleon software (Dionex). Priorto integration, the SEC chromatogram for a buffer containing noetanercept was subtracted from all chromatograms. All integration wasperformed between retention times of 2 minutes and 8 minutes (CHS) or 12and 26 minutes (Innovator). Several parameters were used to define apeak. The minimum area for a detected peak was set to 0.05 mAu*min. Thetwo-dimensional sensitivity for peak detection was set to 0.01 mAu and75 seconds. Peak shoulders were added manually using a manualintegration tool. All detected peaks were manually adjusted in twosteps. First, peak baselines (the bottom boundary of the peak) wereadjusted to horizontal. Secondly, the vertical positions of the peakbaselines were adjusted to that of the chromatogram baseline. Thechromatogram baseline value was defined as the signal in absence ofanalyte. In this case, the signal in absence of analyte was defined asthe absorbance in mAu at 2 minutes retention time.

Hydrophobic Interaction Chromatography (HIC).

Formulation samples were loaded into HPLC vials without dilution beforeinjection on chromatography columns. Samples were separated by HICaccording to the parameters listed in the table below.

TABLE 4 HIC method description Solvent A: 1.8M ammonium sulfate, 0.1Msodium acetate pH 6.0 Solvent B: 0.1M sodium acetate pH 6.0 Column Temp:35° C. Column: TSK-Gel Butyl NPR 14947 Flow Rate: 1.0 mL/min Detection:280 nM Injection: 0.5 uL, 50 mg/mL Enbrel Gradient A time/minutes % B 00 40 50 50 100 50.1 0 55 0

FlowCAM® Method Description for Testing of Formulation 3:5.

Method development work was performed on Jul. 10, 2012 initially using aManuel Prime with Non-Sample procedure (liquid to liquid interphase).Pronounced mixing effects were seen in the flow cell so an alternativeair gap procedure (Manual Prime with Sample) was selected for sampleevaluation.

The baseline (T0) formulation 3:5 sample was received frozen and hadbeen stored at −20° C. until thawed at ambient. Once thawed, theformulation was stored at refrigeration temperature (2-8° C.). The T0sample method included a matching buffer pre-treatment step to conditionthe flow cell. Prior to loading the sample, 0.4 mL or more of thematching formulation buffer was flushed thru the system. It wasdetermined that this pre-treatment was not required and was thereforenot used for the t3 testing time point.

Samples of formulation 3:5 that had undergone three months of heatstress at 5° C. and 25° C. were evaluated in the FlowCAM analysis. Allsamples were thawed on the day they were analyzed. Once thawed, theywere stored at refrigeration temperature (2-8° C.).

Instrumentation & Accessories

-   FlowCAM Instrument: Model VS1, Serial #551 with Sony SX90 camera and    C70 pump with a 1 mL syringe (Fluid Imaging Technologies)-   FlowCAM Software: DSP Firmware Version: 54; version 3.0.3-   Flow Cell Field of View (FOV FC80) with a depth of 80 μm and a width    of 700 μm (Fluid Imaging Technologies)-   Objective: 10×

Context Setting (Method & Setting Parameters)

Method: Manual Prime with Sample (air gap)Sample Analysis: 0.200 mL volume with 0.170 mL analyzedFlow Rate: 0.100 ml/minAuto image rate: 22 frames per second

Efficiency: 38.7%

Run time: 1.7 minutes

Distance to Nearest

Neighbor: 0 micronsClose Hole: 5 iterationsImages: Collage image border padding of 5

Particle Segmentation: Dark Threshold 15.00, Light Threshold 15.00Acceptable Region: Left 15, Right 1255, Top 0, Bottom 959 Camera:Shutter 8 Gain: 57

Auto image rate: 22 frames per secondFlash camera delay: 100 microsecondsFlash duration: 18.5 microsecondsDiameter (ESD): Min 2.00, Max 1000.00 microns

Prior to running samples, the flow cell and objective were installed andthe Field of View and Focus optimization were performed. Systemqualification included running water blanks and particle size standardin multiple replicates. Prior to running samples a cleaning procedurewas undertaken to ensure particle counts were at acceptable levelstypically below 1000 particles/mL between samples or less than 5% of thesample particles/mL between replicate samples. The routine cleaningprocess used water (Millipore Direct-Q type 1, 0.22 μm filtered, 18.2MΩ) between cleaning agents and as a final flush prior to determiningcount levels. Once the particle count reached an acceptable particle permL level the sample was carefully pipetted into the sample tip andloaded into the flow prior to initiating the sample analysis.

Run quality was determined during and immediately after each run using aseries of diagnostic tools in VisualSpreadSheet including x-y captureplot (to visualize flow pattern dynamics), aspectic ratio to diametersize plot (identify stuck particles), image review during the run andimage analysis at completion of run using various particlecharacteristics (e.g. size, circularity, length, aspect ratio).

Individual particle size was determined with Fluid Imaging Technologiessoftware measurement technique known as Equivalent Spherical Diameter(ESD).

ESD is the mean feret measurement of the particle based on 36 samplemeasurements (conducted every 5°). A feret measurement is theperpendicular distance between parallel tangents touching opposite sidesof the particle.

The following data tables describe the behavior of formulation 3:5 overthree months of thermal stress

(Note: In Tables VIII through XIII, number in parentheses (e.g., 3:6)refers to the formulations being tested. The designation “C” is acontrol sample in which 50 mg/ml of etanercept prepared according toExample 4 was present in a formulation consisting of 25 mM phosphatebuffer, 1% sucrose, 100 mM sodium chloride and 25 mM argininehydrochloride

TABLE VIII ONE, TWO AND THREE MONTH STABILITY SEC DATA MONOMER CONTENT(Formulation 3:5 and Comparator) Monomer Content Prior to MonomerMonomer Monomer Monomer Length of Storage/thermal Content at −80° C.Content at 5° C. Content at 25° C. Content at 40° C. Storage stressStorage Temp Storage Temp Storage Temp Storage Temp One Month¹ 99.46(3:5) 99.26 (3:5) 99.17 (3:5) 99.95 (3:5) 83.14 (3:5) 99.55 (C) 99.38(C) 99.37 (C) 96.54 (C) 87.83 (C) One Month² 94.47 (3:5) — 95.31 (3:5)94.06 (3:5)   — (3:5) 95.52 (C) 95.01 (C) 94.30 (C) 84.69 (C) TwoMonths² 94.47 (3:5) — 95.43 (3:5) 91.98 (3:5) 63.84 (3:5) 95.52 (C)95.89 (C) 93.60 68.88 (C) One Month³ 95.08 (3:5) — 94.28 (3:5) 91.86(3:5) 78.04 (3:5) 95.08 94.04 (C) 92.64 (C) 83.96 (C) Two Months³ 95.08(3:5) 93.46 (3:5) 90.78 (3:5) 64.34 (3:5) 95.08 (C) 94.34 (C) 92.22 (C)68.34 (C) Three Months³ 95.08 (3:5) 94.50 (3:5) 90.26 (3:5) 23.63 (3:5)95.08 (C) 94.75 (C) 91.79 (C) 47.46 (C) ¹Sec Method 1; ²SEC Method 2.³SEC Method 3 (Note: In Tables VIII through XIII, number in parentheses(3:5) refers to formulation. The designation “C” is a control sample inwhich 50 mg/ml of etanercept prepared according to Example 4 was presentin a formulation consisting of 25 mM phosphate buffer, 1% sucrose, 100mM sodium chloride and 25 mM arginine hydrochloride.)

TABLE IX ONE, TWO AND THREE MONTH STABILITY SEC DATA-“FRAGMENT 3”(Formulation 3:5 and comparator) Fragment 3 prior to Fragment 3 Fragment3 Fragment 3 Length of storage thermal Content at 5° C. Content at 25°C. Content at 40° C. Storage stress Storage Temp Storage Temp StorageTemp One Month² 3.95 (3:5) 3.29 (3:5) 3.71 (3:5)   — (3:5) 3.27 (C) 3.75(C) 4.12 (C)  7.28 (C) Two Months² 3.95 (3:5) 3.09 (3:5) 4.53 10.39(3:5) 3.27 (C) 3.00 (C) 4.16 (C) 10.76 (C) One Month³ 3.37 (3:5) 4.17(3:5) 5.59 (3:5) — 3.68 (C) 4.57 (C) 5.48 (C) Two Months³ 3.37 (3:5)4.80 (3:5) 5.62 (3:5) — 3.68 (C) 4.22 (C) 5.40 (C) Three Months³ 3.37(3:5) 3.97 (3:5) 5.76 (3:5) — 3.68 (C) 3.84 (C) 5.56 (C) ²SEC Method 2.³SEC Method 3

TABLE X TWO MONTH STABILITY SEC DATA-“AGGREGATE(S)” (Formulation 3:5 andComparator) Aggregates Aggregates Aggregates Length of Content at 5° C.Content at 25° C. Content at 40° C. Storage Storage Temp Storage TempStorage Temp One Month² 0 (3:5) 0 (3:5)   — (3:5) 0 (C) 0 (C)  1.28 (C)Two Months² 0 (3:5) 0 (3:5) 12.28 (3:5) 0 (C) 0 (C)  8.11 (C) ²SECMethod 2.

TABLE XI HIC PEAK 1 (clipped/fragmented species) ONE, TWO AND THREEMONTH STORAGE (Formulation 3:5 and Comparator) PEAK 1 CONTENT PEAK 1PEAK 1 PEAK 1 Prior CONTENT CONTENT CONTENT to Storage/ at 5° C. at 25°C. at 40° C. Length of thermal Storage Storage Storage Storage stressTemp Temp Temp One Month 1.66 (3:5) 1.65 (3:5) 1.91 (3:5) 5.36 (3:5)1.85 (C) 1.71 (C) 1.70 (C) 5.29 (C) Two Months 1.66 (3:5) 1.70 (3:5)3.09 (3:5) 9.27 (3:5) 1.85 (C) 1.78 (C) 2.71 (C) 8.17 (C) Three Months1.66 (3:5) 1.42 (3:5) 2.60 (3:5) 6.46 (3:5) 1.85 (C) 1.42 (C) 2.44 (C)8.05 (C)

TABLE XII HIC PEAK 2 (Etanercept) ONE, TWO AND THREE MONTH STORAGE(Formulation 3:5 and Comparator) PEAK 2 CONTENT PEAK 2 PEAK 2 PEAK 2prior to CONTENT CONTENT CONTENT Storage/ at 5° C. at 25° C. at 40° C.Length of thermal Storage Storage Storage Storage stress Temp Temp TempOne Month 98.34 (3:5) 98.35 (3:5) 98.08 (3:5) 81.45 (3:5) 98.15 (C)98.29 (C) 98.30 (C) 88.39 (C) Two Months 98.34 (3:5) 98.30 (3:5) 96.91(3:5) 69.39 (3:5) 98.15 (C) 98.22 (C) 97.29 (C) 73.93 (C) Three Months98.34 (3:5) 98.98 (3:5) 97.40 26.31 (3:5) 98.15 (C) 98.58 (C) 97.56 (C)54.28 (C)

TABLE XIII HIC PEAK 3 (misfolds/aggregated material) ONE, TWO AND THREEMONTH STORAGE (Formulation 3:5 and comparator) PEAK 3 CONTENT PEAK 3PEAK 3 PEAK 3 Prior to CONTENT CONTENT CONTENT Storage/ at 5° C. at 25°C. at 40° C. Length of thermal Storage Storage Storage Storage stressTemp Temp Temp One Month 0 (3:5) 0 (3:5) 0 (3:5) 13.19 (3:5) 0 (C) 0 (C)0 (C)  6.32 (C) Two Months 0 (3:5) 0 (3:5) 0 (3:5) 21.34 (3:5) 0 (C) 0(C) 0 (C) 17.90 (C) Three Months 0 (3:5) 0 (3:5) 0 (3:5) 67.23 (3:5) 0(C) 0 (C) 0 (C) 37.34 (C)

Formulation 3:5 was evaluated for subvisible particles using the FlowCamflow imaging system. These instruments are designed to measure levels ofsubvisible particles (SVPs). These were measured initially (Table XIV)and then after three months at 5° C. (Table XV) and 25° C. (Table XVI).Consistent with the SEC and HIC data shown above indicating low levelsof aggregate or misfolded material in formulation 3:5 after three monthof thermal stress, formulation 3:5 exhibited low levels of subvisibleparticles (less than 10000 particles per mL having a size greater than 5μm per mL).

In the data presented in Table XIV through XVI, the comparator is thesame as that shown in Tables VIII through XIII above, namely aformulation containing 50 mg/ml etanercept as prepared in Example 4,provided in a formulation comprising 25 mM phosphate buffer, 1% sucrose,100 mM sodium chloride and 25 mM arginine hydrochloride.)

TABLE XIV Initial Number of Particles/mL of Different Sizes as Measuredby FlowCam for Formulation 3.5 (prior to thermal stress) Formulation 3:5Comparator 2-5 μm 14000 ± 16000 7400 ± 8000 5-10 μm 3100 ± 3400 1900 ±1800 10-15 μm 500 ± 500 290 ± 260 15-25 μm 180 ± 120 100 ± 80  25-40 μm130 ± 130 50 ± 40 40-50 μm 20 ± 20 10 ± 10 >50 μm 10 ± 10 10 ± 20 >2 μm17000 ± 21000  9700 ± 11000 >5 μm 3900 ± 4100 2300 ± 2200

TABLE XV Number of particles/mL of Different Sizes as Measured byFlowCam for Formulation 3:5 stored at 5° C. for three months Formulation3:5 Comparator 2-5 μm 38000 ± 18000 7900 ± 4200 5-10 μm 8000 ± 3300 1600± 700  10-15 μm 820 ± 270 150 ± 30  15-25 μm 200 ± 30  40 ± 30 25-40 μm110 ± 40  10 ± 10 40-50 μm 0 ± 0 0 ± 0 >50 μm 10 ± 10 0 ± 0 >2 μm 47000± 22000 9600 ± 4900 >5 μm 9100 ± 3600 1800 ± 700 

TABLE XVI Number of particles/mL of Different Sizes as Measured byFlowCam for Formulation 3:5 stored at 25° C. for three monthsFormulation 3:5 Comparator 2-5 μm 16000 ± 7000  15000 ± 10000 5-10 μm4300 ± 1500 3200 ± 2000 10-15 μm 490 ± 150 310 ± 190 15-25 μm 190 ± 60 170 ± 140 25-40 μm 60 ± 40 50 ± 40 40-50 μm 10 ± 10 10 ± 10 >50 μm 20 ±10 10 ± 10 >2 μm 22000 ± 8600  19000 ± 13000 >5 μm 5000 ± 1700 3800 ±2300

The data presented in Tables VIII through XVI above demonstrate that anamino acid stabilized formulation according to the present invention iscapable of achieving storage stability comparable to or better than thatof a comparator formulation comprising arginine as the stabilizer.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. An aqueous pharmaceutical composition comprisingetanercept and a stabilizer to inhibit instability, aggregation,misfolding and/or fragmentation of the etanercept, wherein thestabilizer comprises one or more amino acid compounds selected from thegroup consisting of serine, proline and glutamate.
 2. The composition ofclaim 1, further comprising one or more additional components selectedfrom: a buffer; a tonicity modifier, and an excipient.
 3. The stabilizedetanercept composition of claim 2 where the stabilizer is glutamate, theformulation is free or essentially free of arginine, and the formulationelicits long term storage stability as characterized by at least one of:SEC analysis at M₃ or T₂ or T₄ of: monomer content greater than about90%; aggregates content of less than about 3 wt %; and fragment 3content less than about 5 wt %: and HIC analysis at M₃ T₂ or T₄ whereinthe amount of the composition represented by peak 1 of the HICchromatogram is less than about 3 wt. %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than 80 wt. %;and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 20 wt. %.
 4. The stabilized etanerceptcomposition of claim 2 wherein the stabilizer is glutamate and theformulation comprises about 25 to about 75 mg/ml etanercept; up to about150 mM glutamate; less than about 6 wt. % sucrose; optionally up toabout 100 mM NaCl; about 1 to about 30 mM sodium phosphate, wherein thecomposition has a pH of about 6.0 to 6.6; and wherein, optionally, thecomposition contains no arginine or is essentially free of arginine. 5.The glutamate stabilized composition of claim 4 eliciting long termstorage stability as characterized by: SEC analysis at M₃ or T₂ or T₄ ofgreater than about 90 wt. % monomer content; less than about 3 wt. %aggregate(s) content; and less than about 5 wt % fragment 3; and HICanalysis at M₃ or T₂ or T₄ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 3 wt.%; the amount of the composition represented by peak 2 of the HICchromatogram is greater than 80 wt. %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 20 wt.%.
 6. The glutamate stabilized composition of claim 5 eliciting longterm storage stability as characterized by: an HIC analysis at M₃ or T₂or T₄ wherein the amount of the composition represented by peak 2 of theHIC chromatogram is greater than or equal to about 95 wt. %; andwherein, if peak 3 is present on the HIC chromatogram, the amount of thecomposition represented by peak 3 is less than or equal to about 3 wt.%.
 7. The glutamate stabilized composition of claim 5 having, at M₃ orT₂ or T₄ no more than, on average, about 10,000 subvisible particles permL having a size greater than 5 μm.
 8. The glutamate stabilizedcomposition of claim 5 comprising about 50 mg/ml etanercept; less thanabout 150 mM glutamate; about 0 to 3% sucrose; about 1 to 30 mMphosphate buffer, optionally up to about 100 mM NaCl, and having a pH ofabout 6.0 to 6.6.
 9. The glutamate stabilized composition of claim 5comprising about 50 mg/ml etanercept; about 100 to about 120 mMglutamate; less than about 4 wt. % sucrose, and about 10 to 30 mMphosphate; and having a pH of about 6.3 to 6.5.
 10. The glutamatestabilized composition of claim 5 comprising about 50 mg/ml etanercept;about 100 mM glutamate; less than about 2 wt. % sucrose, about 100 mMNaCl; about 10-30 mM phosphate; and having a pH of about 6.3 to 6.5. 11.The glutamate stabilized composition of claim 5 comprising about 50mg/ml etanercept; about 50 mM glutamate; less than about 2 wt. %sucrose, about 100 mM NaCl; about 10-30 mM phosphate; and having a pH ofabout 6.3 to 6.5.
 12. The stabilized etanercept composition of claim 2where the stabilizer is serine, the formulation is free or essentiallyfree of arginine, and the formulation elicits long term storagestability as characterized by at least one of: SEC analysis at M₃ or T₂or T₄ of: monomer content greater than about 90%; aggregates content ofless than about 3 wt %; and fragment 3 content less than about 5 wt %:and HIC analysis at M₃ or T₂ or T₄ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 3 wt.%; the amount of the composition represented by peak 2 of the HICchromatogram is greater than 80 wt. %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 20 wt.%.
 13. The stabilized etanercept composition of claim 2 wherein thestabilizer is serine and the formulation comprises up to about 150 mMserine; about 0.5 to about 3 wt. % sucrose; about 1 to about 30 mMsodium phosphate, and wherein the composition has a pH of about 6.0 to6.6; and wherein, optionally, the composition contains no arginine or isessentially free of arginine.
 14. The serine stabilized composition ofclaim 13 eliciting long term storage stability as characterized by: (a)SEC analysis at M₃ or T₂ or T₄ of greater than about 90 wt. % monomercontent; less than about 3 wt. % aggregate(s) content; and less thanabout 5 wt % fragment 3; and (b) an HIC analysis at M₃ or T₂ or T₄wherein the amount of the composition represented by peak 1 of the HICchromatogram is less than about 3 wt. %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than 80 wt. %;and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 20 wt. %; and
 15. The serine stabilizedcomposition of claim 14 which elicits long term storage stability ascharacterized by: an HIC analysis at M₃ or T₂ or T₄ wherein the amountof the composition represented by peak 2 of the HIC chromatogram isgreater than or equal to about 95 wt. %; and wherein, if peak 3 ispresent on the HIC chromatogram, the amount of the compositionrepresented by peak 3 is less than or equal to about 3 wt. %.
 16. Theserine stabilized composition of claim 14 having at M₃ or T₂ or T₄, onaverage, no more than about 10,000 subvisible particles per mL having asize greater than 5 μm.
 17. The serine stabilized composition of claim14 comprising about 50 mg/ml etanercept; about 100-120 mM serine; lessthan about 4 wt. % sucrose, and about 10-30 mM phosphate; and having apH of about 6.3 to 6.5.
 18. The stabilized etanercept composition ofclaim 2 where the stabilizer is proline, the composition is free oressentially free of arginine, and the composition elicits long termstorage stability as characterized by: SEC analysis at M₃ or T₂ or T₄of: monomer content greater than about 90%; aggregates content of lessthan about 3 wt. %; and fragment 3 content less than about 5 wt. %: andHIC analysis at M₃ or T₂ or T₄ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 3 wt.%; the amount of the composition represented by peak 2 of the HICchromatogram is greater than 80 wt. %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 20 wt.%.
 19. The proline stabilized etanercept composition of claim 18comprising up to about 150 mM proline; said composition furthercomprising: about 0.5 to about 3 wt. % sucrose; about 1 to about 30 mMsodium phosphate, about 15 to about 100 mM NaCl; and wherein thecomposition has a pH of about 6.0 to 6.6; and wherein, optionally, thecomposition contains no arginine or is essentially free of arginine. 20.The proline stabilized composition of claim 19 which elicits long termstorage stability as characterized by: SEC analysis at M₃ or T₂ or T₄ ofgreater than about 90 wt. % monomer content; less than about 3 wt. %aggregate(s) content; and less than about 5 wt % fragment 3; and HICanalysis at M₃ or T₂ or T₄ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than about 3 wt.%; the amount of the composition represented by peak 2 of the HICchromatogram is greater than 80 wt. %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 20 wt.%.
 21. The proline stabilized composition of claim 20 which elicits longterm storage stability as characterized by: an HIC analysis at M₃ or T₂or T₄ wherein the amount of the composition represented by peak 2 of theHIC chromatogram is greater than or equal to about 95 wt. %; andwherein, if peak 3 is present on the HIC chromatogram, the amount of thecomposition represented by peak 3 is less than or equal to about 3 wt.%.
 22. The proline stabilized composition of claim 20 having at M₃ or T₂or T₄, on average, no more than about 10,000 subvisible particles per mLhaving a size greater than 5 μm.
 23. The proline stabilized compositionof claim 20 comprising about 50 mM proline, less than about 4 wt. %sucrose, about 25 mM NaCl, about 10-30 mM phosphate, and having pH ofabout 6.3 to 6.5.
 24. The composition of claim 1, containing noarginine, or essentially free of arginine, wherein the composition, atM₃ or T₂ or T₄, elicits long term storage stability that meets one orboth of the following criteria: (A) stability comparable to or betterthan commercially available etanercept marketed under the trademarkEnbrel®, as measured by (i) SEC analysis of the amounts of aggregate(s),monomer and fragment 3 in the composition and (ii) HIC analysis ofamounts of material in the composition corresponding to peaks 1, 2 and 3of the HIC chromatogram; and (B) an HIC chromatogram in which (i) peak 3is absent, or essentially absent and (ii) peak 2 represents greater thanabout 95 wt % of the composition; an SEC chromatogram containingessentially no peak corresponding to aggregate(s); and an SECchromatogram in which the monomer content represents at least about 95wt % of the composition.